本實驗的主要目的是在開發適合臍帶血中造血幹細胞的體外增殖無血清培養基，希望克服臍帶血在臨床移植應用上數量過少的缺點。實驗首先將新生兒的臍帶血進行分離與純化的步驟，可以得到單核細胞(mononuclear cell，MNC)與高均一性的CD133+ cell。接著利用2階因子的實驗設計方法來篩選造血幹細胞體外增殖所需要的細胞激素與血清取代物組成，並搭配著陡升路徑的實驗來找尋細胞激素與血清取代物的最適化濃度，之後再改變各種不同的基礎培養基，最後開發出兩種適合造血幹細胞增殖的無血清培養基，分別為SF-HSC與SF-MNC。SF-HSC的組成為Iscove’s modified Dulbecco’s medium (IMDM)內添加細胞激素配方CC-S6 (8.46 ng/ml TPO、4.09 ng/ml IL-3、15 ng/ml SCF、6.73 ng/ml FL、0.78 ng/ml IL-6 、3.17 ng/ml G-CSF 與1.30 ng/ml GM-CSF)與血清取代物配方BIT2 (1.5 g/l BSA、4.39 μg/ml insulin、60μg/ml transferrin與25.94 μM 2-ME)。CD133+ cell在SF-HSC中批次培養7天可達最佳的增殖效果，white blood cell (WBC)、CD34+ cell、CD34+CD38－ cell、colony-forming unit cell (CFU) cell與long-term culture-initiating cell (LTC-IC)的增殖倍數分別為64.2倍、27.4倍、72.4倍、22.2倍與72.4倍。SF-MNC的組成為IMDM內添加細胞激素配方CC-S9 (5.53 ng/ml TPO、2.03 ng/ml IL-3、16 ng/ml SCF、4.43 ng/ml FL、2.36 ng/ml IL-6 、1.91 ng/ml G-CSF 、1.56 ng/ml GM-CSF、2.64 ng/ml SCGF與0.69 ng/ml IL-11)與血清取代物配方BIT (4 g/l BSA、0.71 μg/ml insulin與27.81μg/ml transferrin)。MNC在SF-MNC中批次培養6天可達最佳的增殖效果，WBC、CD34+ cell、CD34+CD38－ cell、CFU cell與LTC-IC的增殖倍數分別為1.4倍、30.4倍、63.9倍、10.7倍與2.8倍。增殖後的細胞都具有端粒酶活性。
若實驗將培養基的添加策略改為一星期更換兩次，則 WBC與CD34+ cell可以持續的培養達10個禮拜以上，而且細胞的數目都能夠持續的增加。上述的這些培養效果都優於其他的商業培養基，同時實驗所開發的培養基內所含的細胞激素的濃度是其他文獻所提到培養基與商業培養基配方的十分之一，因此證明所開發的SF-HSC與SF-MNC是具有經濟效益與應用潛力的造血幹細胞體外增殖培養基。

The development of ex vivo culture systems that facilitate the expansion of hematopoietic stem and progenitor cells is crucial to stem cell research and clinical application. In this study, two serum-free, stroma-free and cytokine-containing culture media (SF-HSC and SF-MNC) for white blood cells (WBC), CD34+ cells, CD34+CD38－ cells, colony-forming unit cells (CFU cells), and long-term culture-initiating cells (LTC-IC) expansion were systematically developed and optimized using the two-level factorial design and steepest ascent methods. The experimental results showed that the optimal compositions of the serum substitutes and the cytokine cocktail in SF-HSC were BIT2 (1.5 g/L BSA, 4.39 μg/mL insulin, 60 μg/mL transferrin, and 25.94 μM 2-ME), and CC-S6 (8.46 ng/mL TPO, 4.09 ng/mL IL-3, 15 ng/mL SCF, 6.73 ng/mL FL, 0.78 ng/mL IL-6, 3.17 ng/mL G-CSF, and 1.30 ng/mL GM-CSF) in the Iscove’s modified Dulbecco’s medium (IMDM), respectively. After one-week culture, the increases in the total number of WBC, CD34+ cells, CD34+CD38－ cells, CFU cells, and LTC-IC were 64.2-, 27.4-, 72.4-, 22.2-, and 8.1-fold,respectively. The experimental results also showed that the optimal compositions of the serum substitutes and the cytokine cocktail in SF-MNC were BIT (4 g/l BSA, 0.71 μg/ml insulin, and 27.81μg/ml transferring), and CC-S9 (5.53 ng/ml TPO, 2.03 ng/ml IL-3, 16 ng/ml SCF, 4.43 ng/ml FL, 2.36 ng/ml IL-6, 1.91 ng/ml G-CSF, 1.56 ng/ml GM-CSF, 2.64 ng/ml SCGF, and 0.69 ng/ml IL-11) in the IMDM, respectively. After 6-day culture, the increases in the total number of WBC, CD34+ cells, CD34+CD38－ cells, CFU cells, and LTC-IC were 1.4-, 30.4-, 63.9-, 10.7-, and 2.8-fold,respectively.
If SF-HSC or SF-MNC were changed twice per week, WBC and CD34+ cell could grow up continuously for at least 10 weeks. Its expansion ability of CD34+ cells and CFC was superior or comparable to that of X-vivo 20TM, StemlineTM, and StemspanTM commercial media. Furthermore, SF-HSC and SF-MNC has the lowest concentration of cytokine cocktail. These systematic methodologies are helpful in improving the ex vivo expansion system for hematopoietic stem cell and progenitor cells.

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